1. Field of the Invention
The present invention relates to a tissue construct-forming substrate, which is useful for forming a three-dimensional tissue construct containing proliferating cells, a tissue construct-forming kit including the substrate, a method for forming a tissue construct using the kit, and a three-dimensional tissue construct obtained by the method.
2. Description of the Related Art
It has been reported that when case where cultured cells are three-dimensionally assembled to form a tissue construct, the cells survive for a longer period of time and exhibit their original in vivo function at a higher level compared with, for example, a case where cultured cells are two-dimensionally attached to a substrate surface to form a so-called monolayer. Therefore, it is expected that a technique for forming a three-dimensional tissue construct from cultured cells is applied to, for example, a simulator for reproducing biological behavior, an alternative technique for animal experiments in drug development, a regenerative medicine technique by cell transplantation and the like.
As such a technique for forming a three-dimensional tissue construct of cultured cells, a technique in which, for example, primary hepatocytes are cultured in a well which is formed on a substrate and has a cell non-adhesive surface, whereby a tissue construct comprising the primary hepatocytes three-dimensionally assembled in the well has been heretofore proposed (see, for example, Japanese Patent No. 3270286). This document describes that by changing the number of cells inoculated into the well, the size of the tissue construct can be controlled. However, in this technique, in the case where the formed three-dimensional tissue construct contained proliferating cells, the size of the three-dimensional tissue construct continued to increase with the lapse of culture time because the cells proliferated, therefore, it was difficult to control the size of the three-dimensional tissue construct to be constant.
As another method, a technique in which after endothelial cells are two-dimensionally cultured in a plurality of small areas regularly arranged on a surface of a substrate, primary hepatocytes are cultured on a monolayer of the endothelial cells, whereby a tissue construct comprising the primary hepatocytes three-dimensionally assembled on the monolayer is formed has been proposed (see, for example, WO 03/010302). This document describes that in order to prevent a plurality of constructs formed adjacent to one another from adhering to one another, the distance between the plurality of small areas should be set to a predetermined value or more. However, a method for preventing an increase in the size of the three-dimensional tissue construct containing proliferating cells is not considered.
Further, use of a honeycomb porous body formed of a non-water-soluble polymer as a material which can be a favorable scaffold for cell proliferation has been proposed (see, for example, JP-A No. 2001-157574). However, this porous material has a honeycomb structure with a bottom, i.e., a structure in which a plurality of small spaces divided with partitions vertically disposed on a base film are formed on a surface of the base film such that they are positioned in a hexagonal manner, and it is not reported whether or not proliferated cells can maintain their function over a long period of time, or whether or not this honeycomb porous body can be used as a substrate for a cell chip capable of maintaining the function of cells over a long period of time.
On the other hand, in the case where proliferating cells with a high metabolic activity are cultured over a long period of time as a case, for example, where an embryoid body is formed from ES cells, development of a method for forming a tissue construct capable of smoothly performing supply of a fresh culture medium and discharge of waste product or a tissue construct-forming kit capable of realizing the method has been demanded. In general, for example, when a large number of embryoid bodies with a uniform size are formed from ES cells, the hanging drop method is employed (see, for example, Methods in Molecular Biology, vol. 330: Embryonic Stem Cell Protocols: Second Edition vol. 2, (2006) pp. 139-141 (Humana Press)). However, it took time and labor to form a tissue construct by employing this method, and development of an efficient method for forming a three-dimensional tissue construct or a tissue construct-forming kit capable of realizing the method has been also demanded from this viewpoint.